Transcriptional occlusion caused by overlapping promoters.

Abstract

RpoS (σ(38)) is required for cell survival under stress conditions, but it can inhibit growth if produced inappropriately and, consequently, its production and activity are elaborately regulated. Crl, a transcriptional activator that does not bind DNA, enhances RpoS activity by stimulating the interaction between RpoS and the core polymerase. The crl gene has two overlapping promoters, a housekeeping, RpoD- (σ(70)) dependent promoter, and an RpoN (σ(54)) promoter that is strongly up-regulated under nitrogen limitation. However, transcription from the RpoN promoter prevents transcription from the RpoD promoter, and the RpoN-dependent transcript lacks a ribosome-binding site. Thus, activation of the RpoN promoter produces a long noncoding RNA that silences crl gene expression simply by being made. This elegant and economical mechanism, which allows a near-instantaneous reduction in Crl synthesis without the need for transacting regulatory factors, restrains the activity of RpoS to allow faster growth under nitrogen-limiting conditions.

KEYWORDS:

RpoS activity and crl expression under nitrogen-starvation conditions. (A) Induction of dps (Left) and crl (Right) transcription measured under carbon-starvation and nitrogen-starvation conditions relative to nonstarved cells. Error bars indicate the SEM. (B) The crl promoter sequence with both RpoD and RpoN promoters are highlighted. The TSSs are indicated with a +1, and the rbs is indicated in green; asterisks mark the consensus sequence for each of the promoters. (C) Northern blot analysis of crl mRNA in wild-type and ntrC mutant strains. (D) Western analysis of Crl levels in carbon-starved (C-) or nitrogen-starved (N-) and nonstarved cells (LOG).

RpoS activity and crl expression under nitrogen-limiting, M63 (Arg+), conditions. (A) Induction of dps (Left) and crl (Right) transcription measured under nitrogen-limiting conditions relative to nonstarved cells. Error bars represent the SEM. (B) Western blot analysis of Crl and Crl-serine levels in nitrogen-limited and nonlimited cells. Shown is a representative of four independent experiments. Relative differences in Crl levels, as calculated by ImageJ, are shown as percentage of WT. The average difference in Crl levels between the two conditions in the WT strain is threefold.

Transcriptional occlusion at crl. (A) Under nitrogen-rich conditions, the transcript made by the RpoD polymerase (green) is translated well, resulting in high Crl levels. (B) Under nitrogen-limiting conditions the RpoN polymerase (red) acts as a repressor occluding the RpoD polymerase and makes a shorter crl transcript that lacks an rbs, thus inhibiting Crl synthesis and reducing Crl levels.

The effect of the RpoN promoter mutation GC-12AT on crl expression. (A) crl transcription was measured in exponential phase crl null strains carrying plasmid pZS*11crl-1000 with and without the GC-12AT promoter mutation. Data are presented as relative induction over cells carrying the wild-type crl plasmid grown in complete minimal media. Error bars represent the SEM. (B) Western blot analysis of Crl levels in nitrogen-limited and nonlimited cells. Shown is a representative of four independent experiments. Differences in Crl levels, as calculated by ImageJ, are shown as percentage of WT.